The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson\u27s disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check

Mouse chemical mutagenesis to identify new immune mechanisms against cytomegalovirus and herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) and cytomegalovirus (CMV) are ubiquitous and very prevalent pathogens. Infections with these agents result in a variety of clinical syndromes, ranging from asymptomatic to severe infection, with high mortality rates and significant long-term morbidity, particularly in vulnerable in immunocompromised or immature populations. The use of inbred mouse strains that accurately replicate disease and exhibit natural genetic variation in susceptibility has been particularly useful to understand fundamental mechanisms of virus disease pathogenesis. Further, random chemical mutagenesis with N-Ethyl-N-Nitrosourea (ENU) is a powerful tool to identify new mutations on genes and pathways critical to immunity. We have developed an ENU mutagenesis platform followed by a high-throughput infection pipeline allowing us to screen a high number of mice for susceptibility to either CMV or to HSV-1 infection. We generated and tested more than 264 pedigrees, for a total of 4200 animals for gained susceptibility to infection. Doing so, we found 6 families exhibiting susceptibility to HSV-1 and 1 to CMV. We selected one pedigree susceptible to HSV-1 for further analysis. We found a non-synonymous mutation in the linker domain (LD) of STAT5A (W484G). CD8+ T cells and NK cell responses were affected in STAT5AW484G mice, as their numbers were reduced following infection with HSV-1, as were the antigen experienced CD44+ CD8+ T cells. After stimulation with IL-2, which is mediated in part by STAT5A, NK cells did not increase the expression of IL-2Rβ as much as wild type mice, while the expression of IL-2Rα on CD4 and CD8 T cells was abrogated. These results show that STAT5A is important to the T cell and NK cell immune responses to HSV-1 and CMV infection and a single point mutation in its LD is sufficient to compromise STAT5A function. We also analysed a pedigree susceptible to CMV and identified a critical mutation on the uncharacterized GTPase GNL1. NK cells, which play a non-redundant role in CMV resistance, were drastically reduced in mutant animals, causing viral susceptibility. NK cells also exhibited an increase in caspase 3 activity accompanied with a dysfunctional cell cycle progression after IL2 and IL15 stimulation, two cytokines critical for NK cell biology. The inactivation of p53 rescues the NK deficiency and cell death. We showed that GNL1 is crucial for NK cells homeostasis and that GNL1 inactivation leads to NK deficiency and MCMV susceptibility. Taken together, our results allow for a better understanding of the mechanism governing the immune response to herpesviruses and lymphocyte biology, facilitating the elaboration of antiviral and immune treatment.Le virus de l’Herpès Simplex de type 1 (VHS-1) et le Cytomégalovirus (CMV) sont des pathogènes très prévalents. Les symptômes cliniques sont variés, allant d’une infection asymptomatique à de sévères infections pouvant causer la mort ou des effets morbides a long-terme. Les populations immunosupprimées ou au système immunitaire immature sont particulièrement à risque. Afin de comprendre les mécanismes fondamentaux de la pathogenèse, l’utilisation de souche de souris consanguines fut particulièrement utile. Ces différentes souris répliquent les hauts points de l’infection et possède une variation génétique naturelle dans la résistance au virus. De plus, l’introduction aléatoire de mutation chez la souris par l’agent chimique N-Ethyl-N-Nitrosourée (ENU) est un outil puissant afin de découvrir de nouvelles cascades de signalisation et gènes mutés critiques à l’immunité. Dans le cadre de cette thèse, nous avons développé une plateforme de mutagenèse couplée à un pipeline d’infection à haut volume permettant de tester de grandes quantités de souris pour la susceptibilité à CMV et VHS-1. Nous avons généré et infecté plus de 264 pédigrées, pour un total de 4200 animaux. Ce faisant, nous avons trouvé 6 familles susceptibles à VHS-1 et 1 à CMV. Nous avons sélectionné un pédigrée susceptible à VHS-1 pour de plus amples analyses et avons identifié une mutation non synonyme dans le domaine de liaison (DL) de STAT5A (W484G). Les cellules T CD8 et NK étaient affectés par la mutation. Leurs nombres étaient réduits suivant l’infection, tout comme ceux des cellules T CD44+ CD8+ ayant déjà vu l’antigène. Après stimulation avec IL-2, donc le signal est en partie médié par STAT5A, les cellules NK n’ont pas augmenté l’expression du récepteur IL-2Rβ, tandis que l’expression de IL-2Rα était abrogée chez les cellules T CD4 et CD8. Ces résultats démontrent que STAT5A est important pour la réponse immunitaire à VHS-1 et CMV des cellules T et NK, et qu’une mutation ponctuelle dans le DL de STAT5A est suffisant pour compromettre sa fonction. Nous avons aussi sélection un pédigrée susceptible a CMV et identifié une mutation critique dans la GTPase non caractérisée GNL1. Les cellules NK, qui jouent un rôle essentiel et non redondant dans la résistance à CMV, étaient drastiquement réduites, causant la susceptibilité au virus. Le niveau d’activité de caspase 3 était élevé chez les NK, en plus d’avoir un cycle cellulaire dysfonctionnel après stimulation avec IL-2 et IL-15, deux cytokines critiques à la biologie de ces cellules. L’inactivation de p53 restaure un nombre normal de NK et diminue la mort cellulaire. Nous avons démontré que GNL1 est crucial à l’homéostasie des cellules NK et l’inactivation de GNL 1 cause une déficience en cellules NK et la susceptibilité à CMV. Dans l’ensemble, nos résultats permettent une meilleure compréhension des mécanismes régissant la réponse immune aux virus herpétiques et la biologie des lymphocytes, facilitant l’élaboration de traitements antiviraux et immunitaires

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin ▿

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection

Genome-wide mouse mutagenesis reveals CD45-mediated T cell function as critical in protective immunity to HSV-1.

International audienceHerpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc(L3X)), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc(L3X) mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc(L3X) mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc(L3X) mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⁺ and CD8⁺ T cells and could be attributed to function of CD4⁺ T helper 1 (Th1) cells in CD8⁺ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development

ENU-screening strategy.

<p>(<b>A</b>) Schematic representation of the breeding strategy used to produce G3 pedigrees. Details of the breeding procedure are described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003637#s4" target="_blank">Materials and Methods</a> (<i>ENU mutagenesis and breeding</i> section). (<b>B</b>) G3 pedigrees were screened for their susceptibility to HSV-1 infection. Mice were infected i.p. with 1×10⁴ pfu of HSV-1 strain 17 and survival was monitored for two weeks post-infection; susceptible A/J and BALB/c control mice are represented by a blue line, resistant C57BL/6J and C57BL/10J control mice are represented by a green line, all G3 mice by a red line and <i>P43</i> G3s derived from two G2 daughters (G2a and Gb) and one G1 male by brown and black lines. “n” indicates the number of infected mice for each group.</p

CD4⁺, without CD8⁺ T cells, are incapable to protect mice against lethal HSV-1 infection.

<p>(<b>A</b>) Schematic representation of the adoptive transfer strategy. Details of this procedure are described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003637#s4" target="_blank">Materials and Methods</a> (<i>Cell transfer experiments and NK depletion</i> section). (<b>B</b>) <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> mice received 2×10⁷ total splenocytes from either <i>Ptprc^L3X/+</i> or <i>Ptprc^L3X</i> mice. After two hours, these mice were infected i.p. with 1×10⁴ pfu of HSV-1 and their survival was monitored for two weeks (n = 6). (<b>C</b>) <i>Ptprc^L3X/+</i> mice were treated with either anti-asialo GM1 antibody or PBS. After 24 hours, these mice were infected i.p. with 1×10⁴ pfu of HSV-1 and their survival was monitored for two weeks (n = 6). The injection of either anti-asialo GM1 antibody or PBS was performed every three days until the experimental endpoint. (<b>D</b>) <i>Ptprc^L3X</i> mice received either 5×10⁶ T cells or 1.2×10⁷ B cells from <i>Ptprc^L3X/+</i> mice. <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> mice that received only PBS respectively correspond to the positive and negative controls. After two hours, all mice were infected i.p. with 1×10⁴ pfu of HSV-1 and survival was monitored for two weeks (n≥6). (<b>E</b>) <i>Ptprc^L3X</i> mice received either 2.5×10⁶ CD8⁺ or 2.5×10⁶ CD4⁺ T cells from either <i>Ptprc^L3X/+</i> or <i>B6.H2-D^bK^b</i> knock-out (only for the CD4⁺ T cells transfer) mice, whose <i>H2-D^bK^b</i> make them depleted in CD8⁺ T cells (CD8^−/−). <i>Ptprc^L3X/+</i> mice that received only PBS correspond to the positive control. After two hours, all mice were infected i.p. with 1×10⁴ pfu of HSV-1 and survival was monitored for two weeks (n≥6). Data represent two independent experiments. (<b>B–E</b>).</p

The HSV-1 susceptibility associated with the <i>Ptprc^L3X</i> genotype is independent of the infection route and HSV-1 strain.

<p>(<b>A</b>) <i>Ptprc^L3X</i> and heterozygous littermates were infected i.n. with 5×10³ pfu of HSV-1 and monitored for survival. (<b>B and C</b>) <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> mice were infected i.p. with 1×10⁴ pfu of either McIntyre (<b>B</b>) or F strain (<b>C</b>) and monitored for survival. n≥6 for each group and data represent at least two independent experiments. (<b>A, B and C</b>).</p

Expression of viral and inflammatory host genes in brain tissue collected from <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> infected mice.

<p>(<b>A</b>) <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> mice were infected i.p. with 1×10⁴ pfu of HSV-1. At the indicated day (D3, D6 and D10), total brains were harvested and mechanically homogenized for a plaque assay (n≥3). Viral titers are presented as pfu/gram of brain. The dotted line indicates the threshold of detection. (<b>B and C</b>) <i>Ptprc^L3X/+</i> and <i>Ptprc^L3X</i> mice were infected i.p. with 1×10⁴ pfu of HSV-1 (n = 9). Following infection, these mice were weighed two times daily. The brain stems of <i>Ptprc^L3X</i> mice that had lost at least 15% of their pre-infection weight were harvested. <i>Ptprc^L3X/+</i> mice were sacrificed and their brain stems were collected at days 7, 9, and 11 p.i. (n = 3 for each time point). The expression of the <i>ICP4</i> viral gene (<b>B</b>) and the indicated cellular genes (<b>C, upper panels</b>) was normalized to that of <i>hprt</i>. Data are presented as a fold increase relative to infected B6 samples. *, p-value (p)<0.05 and **, p<0.005. Correlations of expression levels were determined by comparing <i>ICP4</i> and the indicated cellular genes (<b>C, lower panels</b>).</p

Identification and characterization of an HSV-1 susceptible ENU mutant.

<p>(<b>A and B</b>) Genome-wide linkage analysis was performed in 45 G3 mice from the <i>P43</i> pedigree (11 susceptible, 34 resistant) using polymorphic markers distinguishing the B6 and B10 genetic backgrounds. (<b>A</b>) Genome-wide linkage quantitative trait loci (qtl) analysis revealed a significant peak on chromosome 1 (LOD score = 6.7, calculated using survival as the phenotype). The horizontal line represents the threshold of significant LOD scores genome-wide, at a p-value = 0.05. (<b>B</b>) Haplotype analysis of the distal portion of chromosome 1 are shown for all G3 mice from the <i>P43</i> pedigree (A, dark gray shading = B6 homozygote; B, white = B10 homozygote; H, light gray = B6/B10 heterozygote; S = susceptible animal; R = resistant animal). (<b>C</b>) Genomic DNA from two G3 <i>P43</i> pedigrees (one resistant and one susceptible) was amplified by polymerase chain reaction and the star indicates the position of the <i>Ptprc^L3X</i> mutation. DNA sequence electropherograms are shown for both resistant and susceptible mice and each of the four nucleotides were labeled with a unique fluorescent dye. Amino acid (aa) positions are displayed in bold; the black arrow indicates the direction of transcription. (<b>D</b>) <i>Ptprc^L3X</i>, <i>Ptprc^L3X/+</i>, <i>Ptprc^+/+</i> and <i>Ptprc^ex9−/−</i> mice were infected i.p. with 1×10⁴ pfu of strain 17 and monitored for survival. “n” indicates the number of infected mice for each group. (<b>E</b>) Blood from <i>Ptprc^+/+</i> and <i>Ptprc^L3X</i> mice was collected and PBMCs were isolated and stained for B220 (n = 3). The expression of B220 was quantified by FACS (upper panel) and represented as a percentage of total cells (lower panel).</p